KRAS Wild-type Colorectal Cancer Research Articles

Combining anti-VEGFR and anti-EGFR antibodies: Randomized phase II study of irinotecan and cetuximab with/without ramucirumab in second-line colorectal cancer:(ECOG-ACRIN E7208).

Early studies showed promise of combined anti-EGFR plus anti-VEGF antibodies for advanced colorectal cancer (CRC), yet this was later rejected as toxic and ineffective in studies not selected for RAS status. We studied advanced KRAS wild-type CRC, as second-line treatment, using irinotecan-cetuximab (IC) with or without the anti-VEGFR antibody, ramucirumab (ICR). Patients with one prior regimen including fluoropyrimidine, oxaliplatin and bevacizumab, with KRAS wild-type tumors, were stratified by ECOG PS, time since last chemotherapy and progression on oxaliplatin, to IC (180 and 500 mg/2 q2w), vs modified ICR (mICR) (150 and 400 mg/m2 plus 6 mg/kg respectively). 102 patients were compared for progression-free survival (PFS) as primary endpoint (85% power for 70% improvement in median PFS from 4.5 to 7.65 months). Of the 102 enrolled, 44 treated with IC and 45 with mICR were evaluable. Median PFS was 6.0 vs 9.2 months respectively (HR 0.75, p = .07, significant by study design for p < .128). Response rate was 23% vs 36% (p = .27) and disease-control rate (DCR) was 52% vs 73% (p = .05). Grade ≥3 toxicity was not equivalent. Overall survival was not significantly different at ∼19 months. Previous phase 3 trials without RAS genotyping, rejected combining anti-EGFR and anti-VEGF drugs. In this randomized multi-center phase 2 study for KRAS wild type CRC (all previously bevacizumab-treated) the addition of ramucirumab, to irinotecan and cetuximab improved PFS and DCR, showing the combination is feasible and effective here. Further phase 3 trials with appropriate patient-selection are required. (NCT01079780).

Identification of KRAS mutation-associated gut microbiota in colorectal cancer and construction of predictive machine learning model.

Gut microbiota has demonstrated an increasingly important role in the onset and development of colorectal cancer (CRC). Nonetheless, the association between gut microbiota and KRAS mutation in CRC remains enigmatic. We conducted 16S rRNA sequencing on stool samples from 94 CRC patients and employed the linear discriminant analysis effect size algorithm to identify distinct gut microbiota between KRAS mutant and KRAS wild-type CRC patients. Transcriptome sequencing data from nine CRC patients were transformed into a matrix of immune infiltrating cells, which was then utilized to explore KRAS mutation-associated biological functions, including Gene Ontology items and Kyoto Encyclopedia of Genes and Genomes pathways. Subsequently, we analyzed the correlations among these KRAS mutation-associated gut microbiota, host immunity, and KRAS mutation-associated biological functions. At last, we developed a predictive random forest (RF) machine learning model to predict the KRAS mutation status in CRC patients, based on the gut microbiota associated with KRAS mutation. We identified a total of 26 differential gut microbiota between both groups. Intriguingly, a significant positive correlation was observed between Bifidobacterium spp. and mast cells, as well as between Bifidobacterium longum and chemokine receptor CX3CR1. Additionally, we also observed a notable negative correlation between Bifidobacterium and GOMF:proteasome binding. The RF model constructed using the KRAS mutation-associated gut microbiota demonstrated qualified efficacy in predicting the KRAS phenotype in CRC. Our study ascertained the presence of 26 KRAS mutation-associated gut microbiota in CRC and speculated that Bifidobacterium may exert an essential role in preventing CRC progression, which appeared to correlate with the upregulation of mast cells and CX3CR1 expression, as well as the downregulation of GOMF:proteasome binding. Furthermore, the RF model constructed on the basis of KRAS mutation-associated gut microbiota exhibited substantial potential in predicting KRAS mutation status in CRC patients.IMPORTANCEGut microbiota has emerged as an essential player in the onset and development of colorectal cancer (CRC). However, the relationship between gut microbiota and KRAS mutation in CRC remains elusive. Our study not only identified a total of 26 gut microbiota associated with KRAS mutation in CRC but also unveiled their significant correlations with tumor-infiltrating immune cells, immune-related genes, and biological pathways (Gene Ontology items and Kyoto Encyclopedia of Genes and Genomes pathways). We speculated that Bifidobacterium may play a crucial role in impeding CRC progression, potentially linked to the upregulation of mast cells and CX3CR1 expression, as well as the downregulation of GOMF:Proteasome binding. Furthermore, based on the KRAS mutation-associated gut microbiota, the RF model exhibited promising potential in the prediction of KRAS mutation status for CRC patients. Overall, the findings of our study offered fresh insights into microbiological research and clinical prediction of KRAS mutation status for CRC patients.

Abstract 6027: 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts

Abstract Background: About 80% of colorectal cancer (CRC) and 50 % of Triple-Negative breast cancer (TNBC) patients overexpress epidermal growth factor receptor (EGFR). Mutations in the KRAS oncogene (present in 40% of mCRC) lead to constitutive over-activation of EGFR and drive de novo resistance to anti-EGFR drugs. Here, we propose to target KRAS-mutant/BRAF mutant EGFR positive mCRC and TNBC using 225Ac-labeled matuzumab, a humanized anti-EGFR monoclonal antibody. Methods: p- -SCN-macropa was used to conjugate matuzumab for radiolabeling with 225Ac and SCN-deferoxamine was used to conjugate matuzumab for labelling with 89Zr. The radioimmunoconjugates were characterized by flow cytometry, HPLC and iTLC. in vitro cytotoxicity was evaluated in EGFR-positive mCRC, TNBC cell lines and 3D spheroids with different levels of EGFR density. Tumor growth was monitored using digital caliper. Mice were treated with either 10 MBq of 89Zr-matuzumab for imaging or three doses of 13 KBq/dose administered of 225Ac-matuzumab 10 days apart. In vivo study endpoint was tumor volume greater than or equal to 1500 mm3. Results: Flow cytometry showed about 95% binding to the cells in all EGFR-positive colorectal cell lines (DLD-1, SW620, SNU-C2B, HT-29) and breast cancer cell lines (MDA-MB-468, MDA-MB-231). in vitro studies showed enhanced cytotoxicity of 225Ac-matuzumab compared with matuzumab. IC50 in the MDA-MB-468 cell line for 225Ac-matuzumab (1.86 ± nM) was 35 times more effective than matuzumab (65.25± 7 nM). Similar trends were observed in the other KRAS-mutant mCRC cell lines and breast cancer cell lines. In 3D spheroid models, the IC50 of 225Ac-matuzumab (3.0 ± 2 nM) in MDA-MB-468, was 20.79 times more effective than matuzumab (62.3 ± 1.38 nM). Similar trends were observed for all the other spheroid models as well. High uptake of 89Zr-matuzumab was observed in both mCRC and TNBC xenografts. 225Ac-matuzumab slowed tumor growth rate in MDA-MB-468 and MDA-MB-231 tumors models compared with control antibody or non-treated controls for 30 days. This study is still ongoing and will last for 2 more months. Conclusion: 225Ac-matuzumab shows very promising outcomes in KRAS-mutant mCRC models and breast cancer models and warrants further investigation. Citation Format: Florence Anjong Tikum, Humphrey Fonge, Fabrice Njotu, Hanan Babeker, Jessica K. Pougoue, Nikita Henning, Alireza Doroudi. 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6027.

Effectiveness of 225Ac-Labeled Anti-EGFR Radioimmunoconjugate in EGFR-Positive Kirsten Rat Sarcoma Viral Oncogene and BRAF Mutant Colorectal Cancer Models.

Eighty percent of colorectal cancers (CRCs) overexpress epidermal growth factor receptor (EGFR). Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 40% of CRCs and drive de novo resistance to anti-EGFR drugs. BRAF oncogene is mutated in 7%-10% of CRCs, with even worse prognosis. We have evaluated the effectiveness of [225Ac]Ac-macropa-nimotuzumab in KRAS mutant and in KRAS wild-type and BRAFV600E mutant EGFR-positive CRC cells invitro and invivo. Anti-CD20 [225Ac]Ac-macropa-rituximab was developed and used as a nonspecific radioimmunoconjugate. Methods: Anti-EGFR antibody nimotuzumab was radiolabeled with 225Ac via an 18-membered macrocyclic chelator p-SCN-macropa. The immunoconjugate was characterized using flow cytometry, radioligand binding assay, and high-performance liquid chromatography, and internalization was studied using live-cell imaging. In vitro cytotoxicity was evaluated in 2-dimensional monolayer EGFR-positive KRAS mutant DLD-1, SW620, and SNU-C2B; in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines; and in 3-dimensional spheroids. Dosimetry was studied in healthy mice. The invivo efficacy of [225Ac]Ac-macropa-nimotuzumab was evaluated in mice bearing DLD-1, SW620, and HT-29 xenografts after treatment with 3 doses of 13 kBq/dose administered 10 d apart. Results: In all cell lines, invitro studies showed enhanced cytotoxicity of [225Ac]Ac-macropa-nimotuzumab compared with nimotuzumab and controls. The inhibitory concentration of 50% in the DLD-1 cell line was 1.8 nM for [225Ac]Ac-macropa-nimotuzumab versus 84.1 nM for nimotuzumab. Similarly, the inhibitory concentration of 50% was up to 79-fold lower for [225Ac]Ac-macropa-nimotuzumab than for nimotuzumab in KRAS mutant SNU-C2B and SW620 and in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines. A similar trend was observed for 3-dimensional spheroids. Internalization peaked 24-48 h after incubation and depended on EGFR expression. In the [225Ac]Ac-macropa-nimotuzumab group, 3 of 7 mice bearing DLD-1 tumors had complete remission. Median survival was 40 and 34 d for mice treated with phosphate-buffered saline and [225Ac]Ac-macropa-rituximab (control), respectively, whereas it was not reached for the [225Ac]Ac-macropa-nimotuzumab group (>90 d). Similarly, median survival of mice bearing HT-29 xenografts was 16 and 12.5 d for those treated with [225Ac]Ac-macropa-rituximab and phosphate-buffered saline, respectively, and was not reached for those treated with [225Ac]Ac-macropa-nimotuzumab (>90 d). One of 7 mice bearing HT-29 xenografts and treated using [225Ac]Ac-macropa-nimotuzumab had complete remission. Compared with untreated mice, [225Ac]Ac-macropa-nimotuzumab more than doubled (16 vs. 41 d) the median survival of mice bearing SW620 xenografts. Conclusion: [225Ac]Ac-macropa-nimotuzumab is effective against KRAS mutant and BRAFV600E mutant CRC models.

KRAS-mutant colorectal cancer cell lines cause a prothrombotic state through the upregulation of thrombin: experimental study

Background: The KRAS genotype status is strongly associated with a prothrombotic state in colorectal cancer, and hypercoagulability and cancer-related thrombosis are among the significant events leading to poor prognosis. However, this correlation has not been confirmed at the cellular level. This study aimed to assess the maximum platelet aggregation rate and thrombin expression induced by colorectal cancer cells under different KRAS genotypes. Materials and methods: Platelet aggregation rate assay and western blotting analysis were used to detect platelet aggregation and thrombin expression induced by four colorectal cancer cells with different KRAS genotypes, including RKO, HCT116, SW480, and SW620. FVIIa/tissue factor and thrombin inhibitors were added to explore changes in platelet aggregation rates induced by colorectal cancer cells and the association between KRAS genotype status and hypercoagulable state. Results: KRAS-mutant cells were more likely to increase maximal platelet aggregation, with RKO, HCT116, SW480, and SW620 inducing 34.7%, 55.4%, 44.4%, and 63.8% of platelet aggregation, respectively. The maximum platelet aggregation rate was higher in the metastatic rectal cancer tumour strain SW620 than in the primary rectal cancer strain SW480. RKO cells had lower thrombin expression than the other three cells. Furthermore, the addition of thrombin inhibitors caused a more significant decrease in the platelet aggregation rate in KRAS-mutant cell lines compared to KRAS wild-type cell lines. Conclusion: Compared to KRAS wild-type colorectal cancer cells, KRAS-mutant colorectal cancer cell lines were more likely to be hypercoagulable through the upregulation of thrombin expression, which was mainly achieved through the TF-thrombin pathway.

Prolonging the stability of cetuximab (Erbitux®) and panitumumab (Vectibix®): An orthogonal assessment of physicochemical, biological and microbiological properties of original opened glass vials and diluted saline preparations

The two anti-epidermal growth factor receptor monoclonal antibodies (mAbs) cetuximab and panitumumab are the pillars for the treatment of EGFR-positive, KRAS wild-type metastatic colorectal cancers. However, stability data of these mAbs are generally missing or incomplete. Here, we report for the first time an orthogonal analysis of the stability of cetuximab (Erbitux®) and panitumumab (Vectibix®), either undiluted vial leftovers or saline dilutions in polyolefin/polyamide infusion bags. All samples were stored at 2–8 °C protected from light, according to their summary of product characteristics (SmPCs). Alternatively, opened vials and preparations were maintained at 25 °C for 15 h, and then stored again at 2–8 °C protected from light to mimic a temporary interruption of the cold chain. Vial leftovers proved stable up to 180 days when stored according to their SmPCs, while compounded preparations in infusion bags maintained their physiochemical, biological and microbiological stability up to 30 days. Additionally, no changes were detected up to 30 days for the same samples undergoing a thermal excursion. Our results provide additional rationale to the SmPCs, crucial especially in the case of reassignment and pre-preparation of bags. This information will allow hospitals to achieve significant cost savings, and better organization of the entire therapeutic process.

Loss of HES1 expression is associated with extracellular matrix remodeling and tumor immune suppression in KRAS mutant colon adenocarcinomas

The loss of HES1, a canonical Notch signaling target, may cooperate with KRAS mutations to remodel the extracellular matrix and to suppress the anti-tumor immune response. While HES1 expression is normal in benign hyperplastic polyps and normal colon tissue, HES1 expression is often lost in sessile serrated adenomas/polyps (SSAs/SSPs) and colorectal cancers (CRCs) such as those right-sided CRCs that commonly harbor BRAF or KRAS mutations. To develop a deeper understanding of interaction between KRAS and HES1 in colorectal carcinogenesis, we selected microsatellite stable (MSS) and KRAS mutant or KRAS wild type CRCs that show aberrant expression of HES1 by immunohistochemistry. By comparing the transcriptional landscapes of microsatellite stable (MSS) CRCs with or without nuclear HES1 expression, we investigated differentially expressed genes and activated pathways. We identified pathways and markers in the extracellular matrix and immune microenvironment that are associated with mutations in KRAS. We found that loss of HES1 expression positively correlated with matrix remodeling and epithelial-mesenchymal transition but negatively correlated with tumor cell proliferation. Furthermore, loss of HES1 expression in KRAS mutant CRCs correlates with a higher M2 macrophage polarization and activation of IL6 and IL10 immunosuppressive signature. Identifying these HES1-related markers may be useful for prognosis stratification and developing treatment for KRAS-mutant CRCs.

Correlations between Demographic, Clinical, and Paraclinical Variables and Outcomes in Patients with KRAS-Mutant or KRAS Wild-Type Metastatic Colorectal Cancer-A Retrospective Study from a Tertiary-Level Center in Romania.

Colorectal cancer (CRC) is a significant global public health concern and its characteristics in Eastern Europe are underexplored. In this retrospective study, data of 225 patients with metastatic colorectal cancer (mCRC) from the Colțea Clinical Hospital's Oncology Department in Bucharest were analyzed between 2015 and 2023. They were divided into two groups based on the presence of KRAS mutation. The primary objective of the study was to investigate whether the presence of KRAS mutations influenced the prognosis of mCRC and to identify any demographic, clinical, or paraclinical factors associated with KRAS mutations in stage IV CRC. The overall survival for the entire study population was 29 months. There was a trend towards increased survival in the KRAS wild-type group (31 months) compared to the KRAS-mutant group (26 months), but this difference did not reach statistical significance. We found that lower levels of education, advanced T stage, advanced N stage, and M1 stage at diagnosis negatively impacted prognosis. Real-world data are crucial in shaping public policy strategies to better support patients with metastatic CRC. Understanding the correlations between the demographic, clinical, and paraclinical variables and the outcomes in mCRC patients with KRAS-mutant and KRAS wild-type colorectal cancer is essential for improving patient care and treatment strategies in Romania and beyond.

Smoking and risk of colorectal cancer according to KRAS and BRAF mutation status in a Japanese prospective Study.

Although smoking is a major modifiable risk factor for many types of cancer, evidence for colorectal cancer is equivocal in Asian populations. Recent Western studies have proposed that the association between smoking and colorectal cancer is restricted to specific tumor molecular subtypes. However, no studies have evaluated the association according to tumor molecular subtypes in Asian populations. In a Japanese prospective population-based cohort study of 18 773 participants, we collected tumor tissues from incident colorectal cancer cases and evaluated KRAS (Kirsten rat sarcoma viral oncogene homolog) and BRAF (v-raf murine sarcoma viral oncogene homolog B) mutation status using target sequencing. Multivariable-adjusted Cox proportional hazard model was used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for associations of smoking with the risk of overall colorectal cancer and its subtypes defined by KRAS and BRAF mutation status. Among 339 cases, KRAS and BRAF mutations were identified in 164 (48.4%) and 16 (4.7%) cases, respectively. The multivariable-adjusted HR for ever smoking compared with never smoking was 1.24 [95% CI: 0.93-1.66], 1.75 [1.14-2.68], 0.87 [0.59-1.29], 1.24 [0.93-1.67] and 1.22 [0.38-3.93] for overall, KRAS wild-type, KRAS-mutated, BRAF wild-type and BRAF-mutated colorectal cancer, respectively. The statistically significant heterogeneity was indicated between KRAS mutation status (Pheterogeneity = 0.01) but not between BRAF mutation status. This study is the first to demonstrate that smokers have an approximately 2-fold higher risk of KRAS wild-type colorectal cancer than never smokers in an Asian population. Our findings support that smoking is a risk factor for colorectal cancer, especially for its subtype without KRAS mutations, in Asian populations.

Platycodin D represses β-catenin to suppress metastasis of cetuximab-treated KRAS wild-type colorectal cancer cells.

Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, is extensively used for clinical therapy in KRAS wild-type colorectal cancer (CRC) patients. However, some patients still cannot get benefit from the therapy, because metastasis and resistance occur frequently after cetuximab treatment. New adjunctive therapy is urgently needed to suppress metastasis of cetuximab-treated CRC cells. In this study, we used two KRAS wild-type CRC cells, HT29 and CaCo2, to investigate whether platycodin D, a triterpenoid saponin isolated from Chinese medicinal herb Platycodon grandifloras, is able to suppress the metastasis of cetuximab-treated CRC. Label-free quantitative proteomics analyses showed that platycodin D but not cetuximab significantly inhibited expression of β-catenin in both CRC cells, and suggested that platycodin D counteracted the inhibition effect of cetuximab on cell adherence and functioned in repressing cell migration and invasion. Western blot results showed that single platycodin D treatment or combined platycodin D and cetuximab enhanced inhibition effects on expressions of key genes in Wnt/β-catenin signaling pathway, including β-catenin, c-Myc, Cyclin D1 and MMP-7, compared to single cetuximab treatment. Scratch wound-healing and transwell assays showed that platycodin D combined with cetuximab suppressed migration and invasion of CRC cells, respectively. Pulmonary metastasis model of HT29 and CaCo2 in nu/nu nude mice consistently showed that combined treatment using platycodin D and cetuximab inhibited metastasis significantly in vivo. Our findings provide a potential strategy to inhibit CRC metastasis during cetuximab therapy by addition of platycodin D.

Variation in FOLFOX, FOLFIRI, and FOLFOXIRI effects on CD8+ T cell and PDL1 levels in MSS CRC patients.

3526 Background: Colorectal cancer (CRC) is a heterogenous disease treated with FOLFOX, FOLFIRI, and FOLFOXIRI chemotherapy regimens. About 85% of CRC patients have microsatellite stable (MSS) tumors that resist immune checkpoint blockade (ICB). Some studies suggest that chemotherapy modulates the MSS CRC tumor microenvironment (TME) to enhance CD8+ T cell infiltration, but ICB remains ineffective. We evaluated the TME of MSS CRC patients to investigate chemotherapy impact on immune markers. Methods: CRC patient samples (n = 16,827) underwent DNA (592-gene or whole exome)/RNA (whole transcriptome) sequencing at Caris Life Sciences. Immune cell fractions within TMEs were estimated from RNA deconvolution (quanTIseq; Finotello, 2019; n = 11,109). PDL1 expression was assessed by IHC (SP142; ≥2+/5%). Patients who received FOLFOX (n = 425), FOLFIRI (n = 88), or FOLFOXIRI (n = 19) &lt; 1 year prior to tumor collection or who didn’t receive these treatments &gt; 4000 days before tumor collection (untreated, n = 6,608) were analyzed. Statistical significance was determined using chi-square, Fisher’s exact, and Mann-Whitney U tests, where appropriate. Results: FOLFOX-treated CRC (n = 213) had a higher CD8+ T cell fraction vs untreated (n = 3,449, FC = 1.9, p &lt; 0.01) CRC and there was no difference among FOLFIRI-treated (n = 37, FC = 1.1, p = 0.13) or FOLFOXIRI-treated (n = 9, FC = 2.2, p = 0.49) CRC. Survival outcomes were similar in FOLFOX-treated CD8+ T cell-high (n = 82) vs low (n = 161, HR = 1.3, p = 0.12) and in FOLFOX-treated PDL1+ (n = 13) vs PDL1- (n = 338) CRC (HR = 1.6, p = 0.14). The CD8+ T cell fraction was similar in untreated KRAS wild-type (WT, n = 1714) vs untreated KRAS-mutated (mut, n = 1718) CRC (FC = 1.3, p &lt; 0.01). FOLFOX-treated KRAS-mut CRC had a higher CD8+ T cell fraction (n = 1718 untreated, 96 treated, FC = 2.3, p &lt; 0.01) and CD69 expression (n = 1724 untreated, 97 treated, FC = 1.7, p &lt; 0.01) vs untreated KRAS-mut CRC. This FOLFOX-dependent effect on CD8+ T cells was lower in WT KRAS CRC (n = 114 FOLFOX-treated, n = 1714 untreated, FC = 1.6, p &lt; 0.01) but survival outcomes were similar in FOLFOX-treated WT (n = 210) vs KRAS-mut (n = 210) CRC (HR = 1.0, p = 0.82). The CD8+ T cell fraction was similar in untreated right- (R; n = 803) vs left-sided CRC (L; n = 927, FC = 1.3, p &lt; 0.01) but PDL1+ IHC rates were significantly higher in R-CRC (n = 1340 L, 1124 R, FC = 2.2, p &lt; 0.01). The CD8+ T cell fraction was similar in untreated WT (n = 3144) vs BRAF-mut CRC (n = 248, FC = 0.8, p &lt; 0.01), but BRAF-mut were more frequently PDL1+ (n = 4622 WT, 340 mut, FC = 4.2, p &lt; 0.01). PDL1+ rates were similar in untreated (n = 1124) vs FOLFIRI-treated R-CRC (n = 12, FC = 2.0, p = 0.46) but were higher in FOLFIRI-treated (n = 9) vs untreated (n = 1340) L-CRC (n = 9, FC = 6.0, p = 0.04). Conclusions: We describe an association between FOLFOX and subsequent infiltration by CD8+ T-cells and high PDL1+ rates in R-CRC, BRAF-mut CRC, and FOLFIRI-treated L-CRC. The findings provide insights for future therapeutic strategies.

Dual role of sprouty2 as an inhibitor of RAS/ERK-driven proliferation and a promoter of cancer invasion in KRAS wild-type colorectal cancer.

Sprouty2 (SPRY2) is known to inhibit the RAS/MAPK/ERK pathway, and is a potential study target for cancer. The effect of SPRY2 in colorectal cancer (CRC) and whether it is influenced by KRAS mutation are not known. We manipulated SPRY2 gene expression and used an activating KRAS-mutant plasmid to determine its effect on CRC cell function in vitro and/or in vivo. We performed SPRY2 immunohistochemical staining in 143 CRC specimens and analyzed the staining results with various clinicopathological characteristics in relation to KRAS mutation status. SPRY2 knockdown in Caco-2 cells carrying the wild-type (WT) KRAS gene upregulated phosphorylated ERK (p-ERK) levels and increased cell proliferation in vitro, but inhibited cell invasion. However, SPRY2 knockdown in SW480 cells (activating KRAS mutant) or Caco-2 cells transfected with KRAS-mutant plasmid did not significantly alter p-ERK levels, cell proliferation, or invasion. The xenografts of SPRY2-knockdown Caco-2 cells were larger with less deep muscle invasion than those of control cells. The clinical cohort study revealed a positive association of SPRY2 protein expression with pT status, lymphovascular invasion, and perineural invasion in KRAS-WT CRCs. However, the associations were not observed in KRAS-mutant CRCs. Interestingly, high SPRY2 expression was related to shorter cancer-specific survival in both KRAS-WT and KRAS-mutant CRC patients. Our study demonstrated the dual role of SPRY2 as an inhibitor of RAS/ERK-driven proliferation and as a promoter of cancer invasion in KRAS-WT CRC. SPRY2 may promote the invasion and progression of KRAS-WT CRC, and might also enhance KRAS-mutant CRC progression through pathways other than invasion.

Abstract 534: PRMT5 inhibition as a therapeutic strategy for KRAS mutant CRC: downstream mediators of the PRMT5-KRAS crosstalk

Abstract Despite decades of research, there are still no effective targeted treatments for the nearly 45% of colorectal cancer (CRC) patients harboring a mutation in their KRAS gene. Protein Arginine Methyltransferase 5 (PRMT5) is an epigenetic regulator undergoing clinical trials as a potential therapeutic target for cancer. Our group has previously reported that PRMT5 is overexpressed in KRAS mutant CRC when compared to KRAS wild-type (WT) CRC, and that PRMT5 inhibition exhibited greater therapeutic efficacy in KRAS mutant CRC when compared to KRAS WT CRC. We therefore proposed that PRMT5 may be a strong therapeutic target for KRAS mutant CRC, and that PRMT5 and KRAS may crosstalk. In this study, we investigated several key downstream signal transduction proteins that may be involved in mediating the potential crosstalk between PRMT5 and KRAS. This is important, as these key intermediate proteins may be strong therapeutic targets for inhibition, as well as valuable therapeutic targets for combination therapy along with PRMT5 inhibitors. We first conducted a literature review to determine which key downstream proteins may be involved in mediating the potential crosstalk between PRMT5 and KRAS. Our initial findings indicated that MYC, p65/RELA, TP53, P21/CDKN1A, ELF1, E2F1, and EIF4E may be key downstream proteins involved in mediating the potential crosstalk between PRMT5 and KRAS. We next used the STRING database to determine which of the aforementioned proteins have demonstrated interactions with PRMT5 and KRAS. We found that PRMT5 and KRAS both interact with MYC, p65/RELA, TP53, P21/CDKN1A, E2F1, and EIF4E with an interaction score &amp;gt; 0.150. We then used the Gene Expression Profiling Interactive Analysis (GEPIA) database to analyze the RNA-Seq data of CRC patients from The Cancer Genome Atlas (TCGA) database. We first used the GEPIA database to determine which proteins are overexpressed in CRC patient tumor samples compared to normal colon and rectum patient samples. We observed that MYC, TP53, E2F1, and EIF4E are all over 2-fold overexpressed in CRC patient tumor samples compared to normal colon and rectum patient samples (q &amp;lt; 0.01). We next used the GEPIA database to determine which proteins have an expressional correlation with PRMT5 and KRAS in CRC patient tumor samples. We found that MYC, E2F1, and EIF4E are all positively correlated with PRMT5 and KRAS with an R-Value &amp;gt; 0.70 and a P-Value &amp;lt; 0.01 in CRC patient tumor samples. Our study thus showcases the key downstream signal transduction proteins that may be involved in mediating the potential crosstalk between PRMT5 and KRAS. These findings are significant, as these key intermediate proteins may be strong therapeutic targets for inhibition, as well as strong therapeutic targets for a PRMT5 combination therapy. Additional research is currently underway to outline the molecular processes behind MYC, E2F1, and EIF4E’s interactions with both PRMT5 and KRAS. Citation Format: David Shifteh, Tzuriel Sapir, Moshe Pahmer, Sanjay Goel, Radhashree Maitra. PRMT5 inhibition as a therapeutic strategy for KRAS mutant CRC: downstream mediators of the PRMT5-KRAS crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 534.

Feasibility of [18F]FSPG PET for Early Response Assessment to Combined Blockade of EGFR and Glutamine Metabolism in Wild-Type KRAS Colorectal Cancer.

Early response assessment is critical for personalizing cancer therapy. Emerging therapeutic regimens with encouraging results in the wild-type (WT) KRAS colorectal cancer (CRC) setting include inhibitors of epidermal growth factor receptor (EGFR) and glutaminolysis. Towards predicting clinical outcome, this preclinical study evaluated non-invasive positron emission tomography (PET) with (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG) in treatment-sensitive and treatment-resistant WT KRAS CRC patient-derived xenografts (PDXs). Tumor-bearing mice were imaged with [18F]FSPG PET before and one week following the initiation of treatment with either EGFR-targeted monoclonal antibody (mAb) therapy, glutaminase inhibitor therapy, or the combination. Imaging was correlated with tumor volume and histology. In PDX that responded to therapy, [18F]FSPG PET was significantly decreased from baseline at 1-week post-therapy, prior to changes in tumor volume. In contrast, [18F]FSPG PET was not decreased in non-responding PDX. These data suggest that [18F]FSPG PET may serve as an early metric of response to EGFR and glutaminase inhibition in the WT KRAS CRC setting.

CircIFNGR2 enhances proliferation and migration of CRC and induces cetuximab resistance by indirectly targeting KRAS via sponging to MiR-30b

Currently the clinical efficacy of colorectal cancer (CRC) which is the most common malignant tumors over the world has not reached an ideal level. Cetuximab, the monoclonal antibody targeting the extracellular domain of EGFR, has shown its great efficacy in the promotion of apoptosis and the inhibition of tumor cells-like characteristics in numerous cancers. However certain KRAS wild-type CRC patients unexpectedly show cetuximab resistance and the specific mechanism remains unclear. Circular RNAs (circRNAs) as the promising novel type of biomarkers in the cancer diagnosis and therapy, have been reported to be related with the drug resistance. In this study, with wondering the mechanism of cetuximab resistance in KRAS wild-type CRC patients, we evaluate the impact of circIFNGR2 on CRC and detect the association among circIFNGR2, miR-30b and KRAS via various experiments such as RT-qPCR, immunohistochemistry, luciferase assays, cell functional experiments and xenograft model. We conclude that circIFNGR2 induces cetuximab resistance in colorectal cancer cells by indirectly regulating target gene KRAS by sponging miR-30b at the post-transcriptional level. It is thus suggested that inhibition of circIFNGR2 can be a promising therapeutic strategy for malignant CRC patients with cetuximab resistance.

Overall survival of patients with metastatic KRAS wild type colorectal cancer patients treated with anti-EGFR third line monotherapy

Introduction. There is no evidence-based data comparing upfront chemotherapy doublets with anti-EGFR monoclonal antibody with sequential treatment utilizing anti-EGFR monotherapy as a consecutive line of treatment in patients with metastatic colorectal cancer. Here we report real-world survival data for patients with colorectal cancer (CRC) treated with anti-EGFR monoclonal antibody as 3rd line monotherapy. Material and methods. It was single center retrospective study. We collected retrospectively data of wild-type KRAS metastatic CRC patients who have failed oxaliplatin- and irinotecan based therapy and were treated with anti-EGFR monoclonal antibody as the 3. line monotherapy in 2009–2017 in Copernicus Memorial Hospital, Lodz, Poland. Last observation was recorded in February 2020. We calculated median overall survival (since commencement of palliative systemic treatment), median progression free survival and median OSIII (overall survival sine commencement of monotherapy with anti-EGFR agent). Results. 130 patients were included in the study. 40.6% were females. The median age was 63 years (range 38–83). 57% of patients were initially diagnosed with metastatic/inoperable colorectal cancer. 80 patients were treated with 3. line cetuximab, 50 — with panitumumab. At the moment of data analysis 123 deaths were recorded. OS since start of palliative systemic treatment was calculated for 120 patients and its median was 25.8 months. MPFS since start of anti-EGFR antibody was 4.3 months, mOSIII —10.7 months. Conclusions. 3rd line treatment of metastatic colorectal cancer with anti-EGFR antibodies is effective. It is good option for patients, who are not fit enough or not willing to have 1st line triplet therapy.

Healthy and unhealthy plant‐based diets in relation to the incidence of colorectal cancer overall and by molecular subtypes

BackgroundPlant‐based foods have been recommended for health. However, not all plant foods are healthy, and little is known about the association between plant‐based diets and specific molecular subtypes of colorectal cancer (CRC). We examined the associations of healthy and unhealthy plant‐based diets with the incidence of CRC and its molecular subtypes.MethodsWhile 123 773 participants of the Nurses’ Health Study and the Health Professionals Follow‐up Study had been followed up (3 143 158 person‐years), 3077 of them had developed CRC. Healthy and unhealthy plant‐based diet indices (hPDI and uPDI, respectively) were calculated using repeated food frequency questionnaire data. We determined the tumoural status of microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and BRAF and KRAS mutations.ResultsHigher hPDI was associated with lower CRC incidence (multivariable hazard ratio [HR] comparing extreme quartiles, 0.86, 95% confidence interval [CI]: 0.77, 0.96; P‐trend = .04), whereas higher uPDI was associated with higher CRC incidence (multivariable HR comparing extreme quartiles, 1.16, 95% CI: 1.04, 1.29; P‐trend = .005). The association of hPDI significantly differed by KRAS status (P‐heterogeneity = .003) but not by other tumour markers. The hPDI was associated with lower incidence of KRAS‐wildtype CRC (multivariable HR comparing extreme quartiles, 0.74, 95% CI: 0.57, 0.96; P‐trend = .004) but not KRAS‐mutant CRC (P‐trend = .22).ConclusionsWhile unhealthy plant‐based diet enriched with refined grains and sugar is associated with higher CRC incidence, healthy plant‐based diet rich in whole grains, fruits and vegetables is associated with lower incidence of CRC, especially KRAS‐wildtype CRC.

Abstract 5138: Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancer

Abstract Background: Recent evidence has highlighted the role of hepatocyte growth factor (HGF) as a putative biomarker to predict EGFR inhibitor resistance. This study investigated the impact of plasma HGF levels on EGFR inhibition and the therapeutic implications of MET inhibition in KRAS, NRAS, and BRAF (RAS/RAF) wild-type colorectal cancer (CRC). Methods: Clinical outcomes were analyzed according to the plasma HGF levels in patients with metastatic CRC (mCRC) receiving palliative first-line cetuximab or bevacizumab + FOLFIRI chemotherapy. Then, in vitro experiments were conducted to validate the clinical findings and to establish pre-clinical evidence of MET inhibition in the HGF-high population. Results: From March 2015 to September 2019, a total of 80 patients were enrolled. The median age was 63 (range, 24-85) years. Of these, 45 patients (56.2%) were treated with bevacizumab + FOLFIRI and 35 patients (43.8%) were treated with cetuximab + FOLFIRI. Among patients with evaluable disease (43 patients (95.6%) in the bevacizumab group and 34 patients (97.1%) in the cetuximab group), the objective response rate was 60.0% and 37.8%, respectively (p=0.162). The median serum HGF level was 374.75 (range, 9.43-30,644.44) pg/mL, and the optimal cut-off value of HGF levels was 12.70 pg/mL by the maximal chi-square method. During a median follow-up of 29.3 (range, 1.2-71.3) months, both progression-free survival (PFS) and overall survival (OS) were significantly shorter in the high HGF group compared with the low HGF group: median 11.8 (95% confidence interval [CI], 10.5-13.1) months vs. 24.7 (95% CI, 23.4-25.9) months for PFS (p=0.009), and median 21.1 (95% CI, 17.9-24.3) months vs. not reached for OS (p=0.018). The difference in PFS and OS was statistically significant in the cetuximab group (p=0.003 for PFS and p=0.035 for OS), but not in the bevacizumab group. In five RAS/RAF wild-type CRC cell lines (Caco-2, COLO 320DM, KM12C, SNU-C1, and SNU-C4), the addition of HGF activated ERK1/2 and AKT via MET phosphorylation. In the cytotoxicity assays, Caco-2 and SNU-C4 were relatively sensitive to cetuximab compared with the others and, in the presence of HGF, cetuximab sensitivity was significantly decreased in the two cells. Moreover, capmatinib, a MET inhibitor, abrogated the effect of HGF on common downstream signaling pathways of EGFR and MET and thus the cetuximab resistance was significantly overcome in vitro. Conclusions: Among patients with mCRC receiving cetuximab + FOLFIRI, those with high plasma HGF levels had significantly worse PFS and OS. HGF induced cetuximab resistance by AKT and ERK activation while capmatinib, a MET inhibitor, significantly increased the anti-tumor effects of cetuximab in the presence of HGF in vitro. Our data may provide evidence of future clinical trials of dual EGFR and MET targeting strategies in patients with HGF-high, RAS/RAF wild-type mCRC. Citation Format: Sang-A Kim, Hyejoo Park, Kui-Jin Kim, Ji-Won Kim, Ji Hea Sung, Milang Nam, Ju Hyun Lee, Eun Hee Jung, Koung Jin Suh, Ji Yun Lee, Se Hyun Kim, Jeong-Ok Lee, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Soo-Mee Bang, Jong Seok Lee, Keun-Wook Lee. Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5138.

A phase 1/2a open label, multicenter study to assess the safety, tolerability, pharmacokinetics, and efficacy of AFM24 in patients with advanced solid cancers: Study design and rationale.

TPS2672 Background: AFM24 is a first-in-class, tetravalent, bispecific, fragment crystallizable-silenced antibody that targets epidermal growth factor receptor-expressing (EGFR+) solid tumors. Of its 4 binding sites, 2 are specific for EGFR, and 2 are specific for CD16A, the Fcγ receptor expressed by natural killer (NK) cells and macrophages. The primary mode of action of AFM24 is not to inhibit EGFR signaling, but to redirect NK cells and macrophages to EGFR+ tumor cells to induce antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), respectively. Preclinical studies showed AFM24 induced killing of EGFR+ tumor cell lines, and the activity was independent of EGFR cell surface expression level. A favorable safety profile was also demonstrated in cynomolgus monkeys. Therefore, AFM24 may utilize the patients’ innate immunity to redirect and activate immune cells, overcoming resistance to current therapies and offering a favorable safety profile. An ongoing Phase 1/2a, first-in-human study (NCT04259450) is evaluating AFM24 in patients with locally advanced or metastatic, treatment refractory solid tumors that are known to express EGFR. The Phase 1 dose escalation study was designed to establish the maximum tolerated dose and/or the recommended Phase 2 dose (RP2D) of AFM24 and evaluated the safety, efficacy, immunogenicity, pharmacokinetic (PK) and pharmacodynamic (PD) responses. AFM24 was administered intravenously once weekly until disease progression, intolerable toxicity, patient withdrawal, or termination at the investigator’s discretion. AFM24 had a well-managed safety profile and the RP2D was established. Methods: In parallel to the continuing dose escalation phase, the Phase 2a dose expansion study was initiated, and the first patient was enrolled in January 2022. This study will assess AFM24 at the RP2D of 480 mg in patients with different EGFR-expressing tumors and will follow a Simon’s two-stage design; the trial will progress to the second stage unless the null hypothesis, that the true tumor response rate is below a specified value, is confirmed at the end of stage one. Eligible patients must have positive histological or cytological staining of EGFR in &gt; 1% of tumor cells. The primary endpoint is to establish the overall response rate (assessed by the investigator per RECIST v1.1) in three disease-specific cohorts. These comprise patients with clear cell renal cell carcinoma (ccRCC), KRAS wild-type colorectal cancer (KRASwt CRC), and EGFR-mutant non-small cell lung cancer (EGFRmut NSCLC). Secondary endpoints include treatment-emergent adverse events, serious adverse events, PK, PD, and immunogenicity. Clinical trial information: NCT04259450.

Genomic landscape of acquired resistance to targeted therapies in patients with solid tumors: A study from the National Center for Precision Medicine (PRISM).

3016 Background: Despite the effectiveness of the various targeted therapies currently approved in solid tumors, acquired resistance remains a persistent problem that limits the ultimate effectiveness of these treatments. Polyclonal resistance to targeted therapy has been described in multiple solid tumors through high throughput analysis of multiple tumor tissue samples from a single patient. However, biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. We used here sequencing of circulating tumor DNA (ctDNA) to characterize the landscape of secondary resistance mechanisms in a large cohort of patients with solid tumors. Methods: This study enrolled patients with advanced cancer from two institutional molecular profiling program STING (NCT04932525, sponsor: Gustave Roussy) or BIP (NCT02534649sponsor: Institut Bergonié). Genomic analysis was performed for each patient by using the Foundation One Liquid CDx Assay (324 genes, tumor mutational burden [TMB], microsatellite instability status). Results: 3435 patients with metastatic disease entered the study. Among them 992 patients (29%) received a targeted therapy matched to a specific molecular alteration before ctDNA. The main tumor types were: prostate cancer (349, 35%), luminal breast cancer (236, 24%), oncogene-addicted non-small cell lung cancer (129, 13%), KRAS-wild type colorectal cancer (126, 13 %). The most frequent class of targeted agents were androgen receptor pathway inhibitor (n = 350, 35%), aromatase inhibitor (236, 24%), anti- EGFR monoclonal antibodies (166, 17%), anti- EGFR tyrosine kinase inhibitors (83, 8%). ctDNA sequencing revealed DNA aberrations involved in secondary resistance in 308 patients (31%). The most frequent aberrations were AR mutations/amplifications, ESR1 point mutations, KRAS point mutations, EGFR point mutations. Among patients with resistance mutation, polyclonal aberrations were identified in 123 patients (40%). The median number of polyclonal aberrations per patient was 2 (range: 2-16). Polyclonal aberrations involved at least 2 different genes in 32 patients (10%). Preliminary results suggest that patients with polyclonal aberrations had worse outcome in comparison with patients with one or no detected aberration and final data will be presented at the time of the congress. Conclusions: We report here the first comprehensive landscape of genomic aberrations in ctDNA involved in resistance to targeted therapies in cancer patients. Polyclonal secondary genomic aberrations represent a frequent clinical resistance mechanism that may explain the poor rate of sustained complete remission observed with targeted therapies and must guide the development of future combinatorial strategies.